Micromechanical Compatibility between Cells and Scaffolds Directs the Phenotypic Transition of Stem Cells.

This study experimentally substantiates that the micromechanical compatibility between cell and substrate is essential for cells to achieve energetically favorable mechanotransduction that directs phenotypic transitions. The argument for this compatibility is based on a thermodynamic model that suggests that the response of cells to their substrate mechanical environment is a consequence of the interchange between forms of energy governing the cell-substrate interaction. Experimental validation for the model has been carried out by investigating the osteogenic differentiation of dental follicle stem cells (DFSCs) seeded on electrospun fibrous scaffolds. Electrospinning of blends containing polycaprolactone (PCL) and silk fibroin (SF) with varying composition of cellulose nanocrystals (CNCs) resulted in three-dimensional (3D) fibrous scaffolds with bimodal distribution of fiber diameter, which provides both macroscopically stiff and microscopically compliant scaffolds for cells without affecting the surface chemical functionality of scaffolds. Atomic force microscopy (AFM) with a colloidal probe and single-cell force spectroscopy were used to characterize cell stiffness and scaffold stiffness on the cellular level, as well as cell-scaffold adhesive interaction (chemical functionality). This study has successfully varied scaffold mechanical properties without affecting their surface chemistry. In vitro tests indicate that the micromechanical compatibility between cells and scaffolds has been significantly correlated with mechanosensitive gene expression markers and osteogenic differentiation markers of DFSCs. The agreement between experimental observations and the thermodynamic model affirms that the cellular response to the mechanical environment, though biological in nature, follows the laws of the energy interchange to achieve its self-regulating behavior. More importantly, this study provides systematic evidence, through extensive and rigorous experimental studies, for the first time that rationalizes that micromechanical compatibility is indeed important to the efficacy of regenerative medicine.

Is the Level of Nitric Oxide in the Dental Follicular Tissues of Impacted Third Molars With a History of Recurrent Pericoronitis a True Marker of Oxidative Stress?

PURPOSE: Nitric oxide (NO) is an indicator of oxidative stress in several tissues. Its role in dental follicular (DF) tissues of impacted third molars with a history of recurrent pericoronitis is not well elucidated. The present study compared NO levels between inflamed and noninflamed DF tissues of impacted third molars with a history of recurrent pericoronitis. MATERIALS AND METHODS: A cross-sectional study was designed. The study sample included inflamed DF tissues (test group) with certain local inflammatory symptoms, such as pain, tenderness, swelling, and erythema and noninflamed DF tissues (control group) without local inflammatory symptoms of impacted mandibular third molars. Each patient contributed only 1 specimen to the samples. All tissues samples were biochemically investigated for NO levels as an indicator of oxidative stress. The primary predictor variable was inflammatory status; secondary predictor variables were age and gender. The primary outcome variable was NO level. Descriptive and comparative analyses were conducted. RESULTS: The test group consisted of 57 patients (28 men, 29 women; mean age, 23.28 ± 5.16 yr) and the control group consisted of 57 patients (30 men, 27 women; mean age, 23.02 ± 5.42 yr). No relevant intergroup differences were noted for demographic findings such as age and gender. NO levels were significantly higher in inflamed DF tissues of impacted third molars than in noninflamed DF tissues (P < .05). CONCLUSION: Results of this study showed that NO might be used as an indicator of oxidative stress and the necessity to remove impacted mandibular third molars with a history of recurrent pericoronitis.

Immunohistochemical analysis of matrix metalloproteinases (1, 2, and 9), Ki-67, and myofibroblasts in keratocystic odontogenic tumors and pericoronal follicles.

BACKGROUND: Keratocystic odontogenic tumor (KCOT) is a benign tumor that arises sporadically or associated with nevoid basal cell carcinoma syndrome (NBCCS). Its locally aggressive behavior contrasts with its cystic histological appearance. To better understand the interaction between tumor cells and the stroma, the present study aimed to evaluate and compare the immunohistochemical expression of matrix metalloproteinases (MMP-1, -2, and -9), the cellular proliferation index (Ki-67), and the presence of myofibroblasts (MFs) in KCOTs. METHODS: Eleven cases of isolated KCOT (G1) and 12 cases of KCOT associated with NBCCS (G2) were selected for an immunohistochemical investigation of the proteins MMP-1, MMP-2, MMP-9, Ki-67, and α-smooth muscle actin (α-SMA) in MFs. A group of 6 pericoronal follicles (G3) was included as a normal odontogenic tissue control. RESULTS: Significant differences between the G3 and G1/G2 groups regarding the expression of MMP-1, MMP-9 (in connective tissue), and Ki-67 were observed. In KCOT, there was a positive correlation between the Ki-67 antigen and MMP-1 and between MFs and MMP-1 in the parenchyma. No statistical differences were found between G1 and G2 groups. CONCLUSIONS: MMP-1, MMP-9, and proliferative activity appear to play important roles in KCOT pathogenesis. The increased proliferative activity with KCOT was associated with elevated MMP-1 production in the parenchyma, which influenced the growth of the lesion in association with an increased number of MFs.

Quantitative and qualitative analysis of epidermal growth factor receptor expression in pericoronal follicles in predicting proliferative potential.

OBJECTIVE: The odontogenic epithelium in pericoronal follicles (PFs) are known to proliferate to form cysts and tumors. This epithelium is mostly composed of the reduced enamel epithelium (REE) and odontogenic rests (OR). The objective of the present study was to evaluate the epidermal growth factor receptor (EGFR) immunoexpression in these PFs to assess their proliferative potential. STUDY DESIGN: The immunoexpression of EGFR in 30 PFs were assessed by two independent observers for intensity, percentage and the location of the EGFR staining. RESULTS: EGFR immunoexpression was noted in 100% of the follicles. A greater proportion of the follicles showed strong intensity (70%). It was noted that nearly 54% of the follicles demonstrated more than 50% of cells with EGFR immunolabelling. EGFR showed combined cytoplasm and membrane staining (40%) and cytoplasm only staining (37%). The analysis of the REE and OR individually for the above-mentioned parameters did not show statistical significance. CONCLUSION: The increased intensity and overall positivity of the epithelium in follicles shows that odontogenic epithelium is responsive to EGFR mediated growth factors. The predominant combined staining pattern is suggestive of increased potential for the epithelium to undergo cystic or neoplastic proliferation.

Immunohistochemical assessment of ATG7, LC3, and p62 in ameloblastomas.

BACKGROUND: To investigate the roles of autophagy in tumorigenesis, cytodifferentiation, and prognosis of odontogenic tumors, we analyzed the immunohistochemical expression of ATG7, LC3, and p62 in odontogenic tissues. METHODS: Tissue specimens of nine dental follicles and 69 ameloblastomas were immunohistochemically examined with antibodies against ATG7, LC3, and p62. RESULTS: Immunohistochemical reactivity for ATG7, LC3, and p62 was detected in many odontogenic epithelial cells and several endothelial cells and fibroblasts in dental follicles and ameloblastomas. ATG7 reactivity in ameloblatomas was significantly higher than that in dental follicles. Expression of ATG7, LC3, and p62 was found markedly in neoplastic cells near the basement membrane rather than central polyhedral cells in ameloblastomas. Reactivity for these molecules was significantly higher in unicystic ameloblastomas than in solid ameloblastomas. Granular cells in granular cell ameloblastomas showed obvious reactivity for the autophagy- related molecules, and LC3 reactivity in granular cell ameloblastomas was significantly higher than in other ameloblastoma variations. Recurrent ameloblastomas showed significantly lower reactivity of LC3 and p62 than primary ameloblastomas. CONCLUSIONS: Expression of ATG7, LC3, and p62 in dental follicles and ameloblastomas suggests that autophagy regulation might be affected by microenvironment alterations during tumorigenesis. The molecular machinery for autophagy is possibly involved in tissue architecture, neoplastic cell differentiation, and prognosis of the benign epithelial odontogenic tumor.

Uniaxially aligned electrospun all-cellulose nanocomposite nanofibers reinforced with cellulose nanocrystals: scaffold for tissue engineering.

Uniaxially aligned cellulose nanofibers with well oriented cellulose nanocrystals (CNCs) embedded were fabricated via electrospinning using a rotating drum as the collector. Scanning electron microscope (SEM) images indicated that most cellulose nanofibers were uniaxially aligned. The incorporation of CNCs into the spinning dope resulted in more uniform morphology of the electrospun cellulose/CNCs nanocomposite nanofibers (ECCNN). Polarized light microscope (PLM) and transmission electron microscope (TEM) showed that CNCs dispersed well in ECCNN nonwovens and achieved considerable orientation along the long axis direction. This unique hierarchical microstructure of ECCNN nonwovens gave rise to remarkable enhancement of their physical properties. By incorporating 20% loading (in weight) of CNCs, the tensile strength and elastic modulus of ECCNN along the fiber alignment direction were increased by 101.7 and 171.6%, respectively. Their thermal stability was significantly improved as well. In addition, the ECCNN nonwovens were assessed as potential scaffold materials for tissue engineering. It was elucidated from MTT tests that the ECCNN were essentially nontoxic to human cells. Cell culture experiments demonstrated that cells could proliferate rapidly not only on the surface but also deep inside the ECCNN. More importantly, the aligned nanofibers of ECCNN exhibited a strong effect on directing cellular organization. This feature made the scaffold particularly useful for various artificial tissues or organs, such as blood vessel, tendon, nerve, and so on, in which cell orientation was crucial for their performance.

Immunohistochemical analysis of CK 18 in dental follicles of impacted third molars: a pilot study.

OBJECTIVE: Odontogenic tumors are lesions derived from epithelial, ectomesenchymal, and/or mesenchymal elements that still are, or have been, part of the tooth-forming apparatus. Approximately 80% of odontogenic tumors occur in the mandible, with a marked predilection for the posterior region, and are often associated with an unerupted tooth. The aim of this study was to determine whether cytokeratin (CK) 18 immunostaining decorated the follicular tissue removed at the time of prophylactic extraction of impacted mandibular third molars, which might suggest oncofetal transformation. MATERIALS AND METHODS: Fifty-four impactions met the study inclusion criteria, of which 24 cases showed the presence of reduced enamel epithelium and/or connective tissue with odontogenic epithelium, which were subjected to CK 18 immunostaining. RESULTS: All 24 cases with adequate epithelium were CK 18 immunonegative. CONCLUSION: There was no oncofetal transformation in the odontogenic epithelia of the dental follicles studied. Thus, although we reaffirm that evaluation of follicular tissue is imperative since disease conditions may be found in minute follicular spaces, development of odontogenic cysts and tumors is unlikely.

Immunohistochemical profile of integrins in enlarged dental follicles and dentigerous cysts.

OBJECTIVE AND STUDY DESIGN: The morphological distinction between incipient dentigerous cyst (IDC) and enlarged pericoronal dental follicle (EDF) remains one of the most controversial questions in the literature. The objective of this study was to analyze the immunohistochemistry expression of alfa(2)beta(1), alfa(3)beta(1), and alfa(5)beta(1) in 23 cases of EDFs and 21 cases of IDCs. RESULTS: All integrins were immunopositive in the cases studied. A significant difference was detected regarding alfa(2)beta(1) integrin (P < .0001) in which a higher expression was present in IDCs. Moreover, statistical difference was also found between basal and suprabasal cell layer in cystic epithelium (P < .0034). The alfa(3)beta(1) integrin expression showed significant difference (P < .013) between EDF and IDC with a tendency of more pronounced staining in IDC. CONCLUSIONS: These results corroborate the possibility of histopathological distinction between EDF and IDC in which squamous metaplasia of reduced enamel epithelium to stratified epithelium would be the first event of cystic transformation.

Epidermal growth factor receptor distribution in pericoronal follicles: relationship with the origin of odontogenic cysts and tumors.

OBJECTIVE: Investigate the distribution of epidermal growth factor receptor (EGFR) in pericoronal follicles as a predictor of progression to odontogenic cysts and tumors. STUDY DESIGN: Immunohistochemical EGFR staining patterns (membrane-only, cytoplasm-only, or combined membrane and cytoplasmic staining) in the reduced enamel epithelium and nests of odontogenic epithelium associated with follicles of impacted molar teeth were evaluated. The staining pattern of 20 specimens of pericoronal follicle was compared with that of 16 normal oral mucosa samples and to squamous cell carcinoma samples. RESULTS: Combined membrane and cytoplasmic staining was observed for normal oral mucosa mostly in proliferating layers (basal and suprabasal), decreasing in intensity toward the surface. Seven epithelial nests presented membrane-only staining, and the majority presented either a cytoplasm-only or a combined staining pattern. The staining patterns observed in reduced enamel epithelium were cytoplasm-only and combined. CONCLUSION: EGFR membrane-only staining may be an indicator of increased potential for epithelial nests to become odontogenic cysts or tumors.

Expression of cell cycle regulatory proteins in epithelial components of dental follicles.

Dental follicle is a component of tooth germs, which remain adjacent to the crown of unerupted or impacted teeth. Under the influence of pathologic changes, however, dental follicles that possess reduced epithelium can proliferate into stratified squamous epithelium as far as originate dental cysts. In order to clarify the role of apoptosis and cellular proliferation herein, expression of p53 and PCNA was examined in epithelial components of dental follicles associated with impacted third molars by means of immunohistochemistry. A total of 40 cases was included in this study being 22 cases with reduced epithelium and 18 cases with stratified epithelium. Expression of p53 expression was weak or not detected in dental follicles with reduced and stratified squamous epithelium. By contrast, PCNA positive cells were evidenced in basal and supra basal layers of the stratified squamous epithelium and in reduced epithelium of dental follicles, but without any significant statistically differences between them (P > 0.05). In conclusion, these data suggest that dental follicles possess proliferative activity as depicted by PCNA-positive nuclei in some epithelial cells. However, the biological behavior of dental follicles during the late stage of dental eruptive process may not be associated with deregulation of death and/or cell proliferation.

Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.

[Establishment of cultivating technology of dental follicle cells of rat and their identifying].

OBJECTIVE: To establish a method for culturing dental follicle cells of rat and observe the growth characteristics of the cultured cells in vitro. METHODS: Dental follicle and associated enamel organs were dissected from the first and second mandibular molars of 6-7-day-old rats, and then cultured in vitro. Purified dental follicle cells derived from the third or the fourth passage cells were utilized in the following experiments. The shape and ultrastructure of dental follicle cells were observed by light-microscopy and transmission electron microscopy. Immunocytochemistry was used to detect the expression of vimentin, type I collagen and fibronectin. RESULTS: The cultured cells were fibroblastic in shape, elongate with processes, and transmission electron microscopy revealed that they contained electron-dense granules, an abundant rough endoplasmic reticulum, but did not form desmosomes. Vimentin, type I collagen and fibronectin were present in dental follicle cell. CONCLUSION: The dental follicle cells of rat could be successfully cultured in vitro and the cultured cells had the same characteristics of dental follicle cells of normal rat.

[Colony-stimulating factor-1 receptor in rat dental follicle cells].

OBJECTIVE: To study localization and expression of CSF-1 receptor protein, in order to discover the CSF-1 and IL-1alpha effects on CSF-1 receptor mRNA levels and to determine if the autocrine effect is inhibited through the CSF-1 receptor. METHODS: Immunolocalization of CSF-1 receptor in the cultured dental follicle cells and in mandibles of the post-natal rats from day 1 to 11 were performed. The effects of different concentrations of CSF-1, IL-1alpha on CSF-1 receptor gene expression were detected by means of RT-PCR. RESULTS: Cultured dental follicle cells were immunostained for the CSF-1 receptor. In vivo, immunostaining showed that the CSF-1 receptor was present in the dental follicle of the first mandibular molar at early post-natally and was either absent or greatly reduced by day 11 post-natally. High concentrations of cvCSF-1 reduced the gene expression of the CSF-1 receptor. IL-1alpha had no effects on CSF-1 receptor mRNA levels. CONCLUSIONS: The expression of CSF-1 receptor reaches a peak early post-natally in the dental follicle of the first mandibular molar of the rat and then subsequently declines. High concentrations of CSF-1 inhibits the expression of CSF-1 receptor, IL-1alpha has no effect on the expression of CSF-1 receptor mRNA.

Synthesis and secretion of MCP-1 by dental follicle cells--implications for tooth eruption.

The monocyte chemotactic protein-1 (MCP-1) gene is expressed in the dental follicle, a loose connective tissue sac that must be present for eruption to occur. The role of MCP-1 may be to recruit mononuclear cells (monocytes) to the dental follicle, where these cells, in turn, fuse to form osteoclasts to resorb alveolar bone for the formation of an eruption pathway. Thus, it was the aim of this study to determine if MCP-1 is secreted by dental follicle cells in culture and if its secretion is enhanced by potential tooth eruption molecules. Western blotting and a two-site capture enzyme-linked immunoabsorbent assay demonstrated that MCP-1 was synthesized and secreted into the medium by the follicle cells. Incubation of the cells with either transforming growth factor-beta one (TGF-beta 1) or interleukin-one alpha (IL-1 alpha) enhanced the secretion of MCP-1 by the cells. Measurement of the chemotactic ability of the conditioned medium to attract mouse monocytes demonstrated that the chemotaxis of the medium was increased if the cells had previously been incubated in IL-1 alpha, although there appears to be a threshold concentration of MCP-1 above which chemotaxis is not enhanced. These combined results suggest that the critical initial cellular event of tooth eruption, an influx of mononuclear cells into the dental follicle at an early post-natal age, may be initiated by the secretion of MCP-1 by the dental follicle cells.

Transcription and translation of CSF-1 in the dental follicle.

The dental follicle, a loose connective tissue sac which surrounds the unerupted tooth, is required for eruption to occur. Injection of colony-stimulating factor-1 (CSF-1) will accelerate molar eruption in rats, as well as stimulate tooth eruption in osteopetrotic rats. Utilizing in situ hybridization and reverse- transcription polymerase chain-reaction techniques, we show here that CSF-1 mRNA is present in vivo in the dental follicle of the first mandibular molar of the rat. Analysis of the molars from day 0 through day 10 post-natally demonstrates that the maximal expression of CSF-1 mRNA is at day 3 post-natally. Immunostaining also reveals that the CSF-1 mRNA is translated, with immunostaining for the CSF-1 itself, being heavy in early post-natal days and absent by day 9 postnatally. In view of the fact that there is a maximal influx of mononuclear cells (monocytes) into the dental follicle at day 3 post-natally--an influx which increases the numbers of osteoclasts needed to form a tooth eruption pathway--it is probable that the maximal expression of CSF-1 mRNA by day 3 post-natally contributes to this monocyte influx. Thus, this study establishes a relationship among a molecule (CSF-1), cell (monocyte), and tissue (dental follicle) that appear to play a major role in tooth eruption.

S100, alpha-smooth muscle actin and cytokeratin 19 immunohistochemistry in odontogenic and soft tissue myxomas.

AIMS: To compare the expression of S100 protein, alpha-smooth muscle actin (alpha-SMA) and keratin 19 in odontogenic myxomas and non-odontogenic myxoid lesions. METHODS: Formalin fixed, paraffin wax embedded tissue from seven odontogenic myxomas, three soft tissue myxomas, six hyperplastic myxoid dental follicles, two intramuscular myxomas, 12 cardiac myxomas, and seven normal dental follicles were examined immunocytochemically for S100 protein, alpha-SMA and cytokeratin 19 using the Streptavidin-biotin method. RESULTS: A minority of odontogenic myxomas (three of seven) were positive for S100 and the staining was of moderate intensity and in all myxofibroblasts. Soft tissue myxomas, normal dental follicles, intramuscular myxomas, and most enlarged myxoid follicles were negative. In the cardiac myxomas the cells forming cords and islands were positive in approximately half (seven of 12), but the dispersed stellate myxoblasts were positive in only two cases. A population of cells in all the odontogenic myxomas and hyperplastic dental follicles contained alpha-SMA, but such cells were sparse in cardiac myxomas and present in only four cases. Cytokeratin 19 was present in odontogenic epithelium of odontogenic myxoma and follicles. CONCLUSIONS: A minority of odontogenic myxomas, but not other oral myxoid lesions, may express S100 protein and this could cause difficulty distinguishing myxoma from myxoid nerve sheath tumours. Sparse myofibroblastic cells occurred in all types of myxoma tested. The epithelium sometimes found within jaw myxomas expresses cytokeratin 19 and this is consistent with an odontogenic origin.

Estudo imunocitoquímico comparativo dos folículos pericoronários, cistos dentígeros e queratocistos odontogênicos / Immunohistochemical comparative study of the dental follicles with dentigerous cysts and odontogenic keratocysts

Com o objetivo de estabelecer as características imunocitoquímicas de folículos pericoronários, cistos dentígeros e queratocistos odontogênicos, foram selecionados dos arquivos do Laboratório de Anatomia Patológica do Departamento de Patologia da Faculdade de Odontologia de Bauru USP, dez casos de folículos pericoronários com epitélio reduzido do orgäo do esmalte, dez casos de folículos pericoronários com metaplasia escamosa, dez casos de cistos dentígeros e dez casos de queratocistos odontogênicos, submetidos a evidenciaçäo imunocitoquímica de um painel constituído dos seguintes marcadores: citoqueratina de alto peso molecular, citoqueratina de baixo peso molecular, laminina, fibronectina, colágeno IV, vimentina e proteína S100. A partir dos resultados obtidos pudemos concluir que o padräo imunocitoquímico das 4 condiçöes estudadas permite uma diferenciaçäo diagnóstica entre folículos pericoronários, cistos dentígeros e queratocistos odontogênicos, utilizando-se a marcaçäo da proteína S100. A diferenciaçäo pela marcaçäo com a proteína S100 pode ser complementada pela evidenciaçäo das células de Langerhans, que caracteristicamente estäo presentes nos revestimentos epiteliais dos cistos dentígeros

A chondroitin sulfate epitope in mammalian dental pulp and its developmental expression in mouse dental papilla.

The molecular specificity of the dental papilla of a bell-stage tooth was studied by production of dental-papilla-reactive monoclonal antibodies (Mabs). One of the Mabs, designated 7C5, recognized an epitope present in glycosaminoglycan. Several lines of evidence suggested that the 7C5-epitope consists of chondroitin 6-sulfate. The Mab did not react with mouse dental epithelium, but reacted uniformly with mesenchymal tissue in the mandibular process and accumulated in the dental sac and in the papilla of bell-stage tooth germs. The 7C5-staining was lost from the differentiating odontoblasts, while the staining in the molar tooth papilla was accumulated in the subodontoblastic layer. In the developing mouse incisor, the 7C5-epitope was restricted to the lingual-posterior area. The 7C5-epitope was also present in pulpal tissue and predentin of different types of teeth of various mammalian species, including man, sheep, swine, and rat. Collagenase pre-treatment of tissue sections abolished the bulk of the 7C5-reactivity in peridental mesenchyme during embryonic stages while leaving the staining of the dental papilla intact. In newborn and adult teeth, collagenase also impaired the reactivity in the pulp except for the subodontoblastic layer. This suggests the existence of different subpopulations of the 7C5-epitope containing proteoglycans in dental papilla and pulp. A high-molecular-weight proteoglycan, sensitive to chondroitinase ABC but not to heparinase or heparitinase, was immunoprecipitated by 7C5 from extracts of bell-stage mouse tooth germs. We suggest that the evolutionary conservation of chondroitin 6-sulfate in the dental pulp reflects its properties as non-terminally differentiated tissue and perhaps the retention of a potential to differentiate to odontoblasts.

Isolation of granule proteins from cells of the dental follicle and stellate reticulum of rat mandibular molars.

The presence of a dental follicle is required for eruption of teeth of limited eruption but it is uncertain if any molecules indigenous to the follicle regulate this eruption. However, electron-dense granules of unknown composition and function are present in the fibroblasts of the dental follicle of rat molars, as well as the adjacent stellate reticulum, before and during tooth eruption. Here the granules have been isolated; two proteins, of 167 and 200 kDa, have been determined by biochemical and immunological methods to be major components of the granules.

Granule proteins of the dental follicle and stellate reticulum inhibit tooth eruption and eyelid opening in postnatal rats.

Electron-dense granules within cells of the dental follicle and stellate reticulum of rat mandibular molars can be isolated; their major components are 167 and 200 kDa proteins. Injecting these granule proteins into postnatal rats results in a delay of incisor eruption and eyelid separation. These inhibitory effects were most pronounced with the 167 kDa protein (a delay of 3 days in incisor eruption and of 2 days in eyelid opening) and were opposite to the stimulatory effects of epidermal growth factor. Thus, these granules may play an inhibitory part in tooth eruption.